Molecular regulation of fistula adaptation for dialysis access

描述(由申请人提供)： 终末期肾病(ESRD)困扰着美国50多万人的生活，每年新增病例超过10万例，死亡率约为每年8.8万人。退伍军人事务部照顾大约30,000名患有ESRD的退伍军人。治疗终末期肾病的费用在2008年约为390亿美元，继续增长。肾脏替代治疗最常见的选择是血液透析；血液透析的途径是动静脉瘘(AVF)、动静脉移植物(AVG)或临时放置导管。尽管瘘管需要时间来“成熟”，例如扩张、增厚和增加流量，但在开始透析之前，动静脉瘘仍然是首选的通路模式，与动静脉瘘和导管通路相比具有更好的长期效果。尽管AVF接入与其替代方案相比具有优越性，但AVF仍远未达到完美。大约20%-50%的动静脉动静脉瘘不成熟，其中许多动静脉动静脉瘘需要一些治疗干预才能成功成熟。事实上，这些糟糕的临床结果表明，AVF违反了血管外科的所有规则；尽管AVF具有长度短、直径大、流量大和阻力小的特点，但在血管外科医生进行的任何手术中，AVF表现出最差的通畅性。动静脉瘘通畅性差清楚地反映了我们对导致动静脉瘘成熟的静脉重塑生物学的不完全了解，以及对增强动静脉动静脉瘘成熟和随后的瘘管使用的新方法的医学需求未得到满足。最近，一些EPhin-Eph途径的成员被描述为发育特定的血管识别的关键决定因素，其中EPhin-B2配体是动脉的胚胎决定因素，Eph-B4受体是静脉的胚胎决定因素。然而，目前尚不清楚Eph-B4在成人静脉中是否具有功能，以及EPhin-Eph通路是否在AVF适应动脉环境过程中发挥机械作用。我们推测，在正常的AVF成熟过程中，Eph-B4的表达和/或激活(通过磷酸化评估)介导了AVF管壁直径的增加和增厚。这是一种不同于Eph-B4介导静脉移植适应的机制。我们将通过以下目的来验证我们的假说：目的I：确定Eph-B4是否在正常的动静脉瘘成熟过程中介导管径扩张和/或管壁增厚。我们的假设是，基于我们的初步数据，静脉规范基因Eph-B4的表达和/或激活增加导致正常的AVF直径扩张、管壁增厚和瘘管成熟。我们将检查来自接受手术翻修或结扎的专利人类AVF的外科标本，以确定AVF直径和/或壁厚如何与Eph-B4的表达和激活相关。我们将通过在我们的小鼠AVF模型中直接增加和减少Eph-B4信号来验证这一假设，并将血管特性与AVF直径和壁厚相关联。目的II：确定切应力如何调节Eph-B4的磷酸化和表达。我们的假设是，切应力通过刺激内皮细胞中Eph-B4信号和表达来促进AVF的成熟；通过这种方式，切应力的变化导致AVF扩张和管壁增厚。我们将在我们的体外血流模型中通过改变剪应力来验证这一假设，使用全静脉和内皮细胞流动模型，并证明动脉剪应力的大小刺激Eph-B4的磷酸化和表达。目的III：研究Eph-B4信号对血管细胞功能的调节作用。我们的假设是，酪氨酸-774上的Eph-B4磷酸化是血管细胞中Eph-B4下游功能的关键调节因子。我们将通过检测野生型和突变型Eph-B4对内皮细胞和平滑肌细胞信号转导以及细胞增殖和迁移的不同影响来检验这一假说。

DESCRIPTION (provided by applicant): End-stage renal disease (ESRD) upsets the lives of over half a million people in the United States, with an incidence of over 100,000 new cases per year and a mortality rate of approximately 88,000 people each year. The Veteran's Affairs cares for approximately 30,000 veterans with ESRD. The costs to treat ESRD, approximately $39 billion in 2008, continue to grow. The most common option chosen for renal replacement therapy is hemodialysis; access for hemodialysis is performed by either arteriovenous fistula (AVF), arteriovenous graft (AVG), or temporary catheter placement. Although fistulae take time to "mature," e.g. dilate, thicken and increase flow, prior to beginning dialysis, AVF continue to be the preferred mode of access, with superior long term results compared to AVG and catheter access. Despite the superiority of AVF access compared to its alternatives, AVF are still far from perfect. AVF fail to mature in approximately 20-50% of cases, with many of these AVF requiring some therapeutic intervention to mature successfully. In fact, these poor clinical results demonstrate that AVF defy all the rules of vascular surgery; despite their desirable characteristics of short length, lage diameter, high flow, and low resistance runoff, AVF exhibit the worst patency of any procedure performed by vascular surgeons. The poor patency of AVF clearly reflects our imperfect understanding of the biology of venous remodeling leading to AVF maturation and the unmet medical need for novel approaches to enhance AVF maturation and subsequent fistula usage. Several members of the Ephrin-Eph pathway have recently been described as developmentally specified critical determinants of vessel identity, with Ephrin-B2 ligand an embryonic determinant of arteries and Eph-B4 receptor an embryonic determinant of veins. However, it is currently not established whether Eph- B4 is functional in adult veins, and whether the Ephrin-Eph pathway plays a mechanistic role during AVF adaptation to the arterial environment. We hypothesize that increased Eph-B4 expression and/or activation (as assessed by phosphorylation) mediates increased AVF wall diameter and thickening during normal AVF maturation. This is a different mechanism than by which Eph-B4 mediates vein graft adaptation. We will test our hypothesis with these aims: Aim I: To determine whether Eph-B4 mediates diameter expansion and/or wall thickening during normal AVF maturation. Our hypothesis, based on our preliminary data, is that increased expression and/or activation of the venous specification gene Eph-B4 leads to normal AVF diameter expansion and wall thickening and fistula maturation. We will examine surgical specimens derived from patent human AVF undergoing surgical revision or ligation to determine how AVF diameter and/or wall thickness correlate with Eph-B4 expression and activation. We will test this hypothesis by directly increasing and decreasing Eph-B4 signaling in our mouse AVF model and correlating vessel identity with AVF diameter and wall thickness. Aim II: To determine how shear stress regulates Eph-B4 phosphorylation and expression. Our hypothesis is that shear stress promotes AVF maturation by stimulating Eph-B4 signaling and expression in endothelial cells; in this way, changes in shear stress lead to AVF dilation and wall thickening. We will test this hypothesis by altering shear stress in our in vitro flow models, using both whole vein and endothelial cell flow models, and show that arterial magnitudes of shear stress stimulate Eph-B4 phosphorylation and expression. Aim III: To determine how Eph-B4 signaling regulates vascular cell function. Our hypothesis is that Eph- B4 phosphorylation on tyrosine-774 is a critical regulator of Eph-B4 downstream function in vascular cells. We will test this hypothesis by examining the differential effects of wild type and mutated Eph-B4 on endothelial and smooth muscle cell signal transduction as well as cell proliferation and migration.